LID scientists are collaborating with scientists from MedImmune under a CRADA to generate candidate vaccines against avian influenza viruses of each subtype, including H5N1 viruses that have caused human infections since 2003. The vaccines were generated using plasmid based reverse genetics and each contains the hemagglutinin and neuraminidase genes from an avian influenza virus and six internal gene segments from the AA ca virus. Based on promising preclinical data in mice and ferrets, clinical lots of vaccines were generated and Phase I clinical trials of the safety and immunogenicity of the vaccines for healthy adults were undertaken under an IND. Several factors contribute to the possibility of an H2 influenza pandemic. First, H2 viruses have proven capable of causing disease in humans. Of the sixteen HA and nine NA subtypes, only H1, H2 and H3 viruses have been known to lead to human influenza pandemics since influenza viruses were first isolated in 1933. While H1N1 and H3N2 viruses have been co-circulating for the past 30 years, H2N2 viruses have not circulated in humans since 1968. Thus, there is declining pre-existing immunity to H2 influenza viruses in the population today. Although H2 influenza has not circulated in humans since 1968, this subtype is maintained in avian reservoirs worldwide. The cold adapted (ca) influenza A/AA/6/60 (AA) (H2N2) virus is the backbone for the live attenuated trivalent seasonal influenza vaccine licensed in the United States. In addition to evaluating the AA ca virus as an H2 subtype vaccine, we selected a group of geographically and temporally diverse wild-type H2 influenza viruses and evaluated the kinetics of replication and compared the ability of these viruses to induce a broadly cross-reactive antibody response in mice and ferrets. In both mice and ferrets, A/Japan/305/1957 (H2N2), A/mallard/NY/1978 (H2N2) and A/swine/MO/2006 (H2N3) elicited the broadest cross-reactive antibody responses against heterologous H2 influenza viruses as measured by hemagglutination-inhibition and microneutralization assays. These data suggested that these 3 viruses may be suitable candidates for development as live attenuated H2 pandemic influenza vaccines. In collaboration with scientists at Yale University, we had previously generated an efficacious vesicular stomatitis virus (VSV)-based AIV vaccine expressing H5 hemagglutinin (HA) from the fifth genomic position of VSV. We characterized VSV-based vaccines that express the HK/156 (clade 0) H5 HA from the first position of the VSV genome. These vectors induce broadly cross-neutralizing antibodies against homologous and heterologous H5N1 viruses of different clades in mice. The vaccines provided complete protection against morbidity and mortality after heterologous challenge with clade 0 and clade 1 H5N1 strains in animals even 1 year after vaccination. Post-challenge pulmonary virus loads showed that these vectors provide sterilizing immunity. Therefore, VSV-based AIV vaccines are potent, broadly cross-protective pandemic vaccine candidates. Intramuscular administration of inactivated influenza vaccine is the main vaccine platform used for the prevention of seasonal influenza infection. In clinical trials, inactivated H5N1 vaccines have been shown to be safe and capable of eliciting immune correlates of protection. However, the H5N1 vaccines are poorly immunogenic compared to seasonal influenza vaccines. Needle-free vaccination would be more efficient and economical in a pandemic and developing an effective and safe mucosal adjuvant will be an important milestone. A stabilized chemical analog of double-stranded ribonucleic acid, PIKA is a potent mucosal adjuvant in a murine model. While PIKA stimulates dendritic cells in vitro, little was known about its receptor and adjuvanting mechanism in vivo. In collaboration with scientists in Singapore, we demonstrated that the immuno-stimulatory effect of PIKA resulted in an increased number of mature antigen-presenting cells, with the induction of pro-inflammatory cytokines at the inoculation site. In addition, co-administration of PIKA with a poorly immunogenic subunit H5N1 vaccine led to antigen sparing and quantitative and qualitative improvement of the immune responses over those achieved with an unadjuvanted vaccine in mice. The adjuvanted vaccine provided protection against lethal challenge with homologous and heterologous H5N1 wildtype viruses. Mice lacking functional TLR3 showed diminished cytokine production with PIKA stimulation, diminished antibody responses and reduced protective efficacy against wildtype virus challenge following vaccination. These data suggest that TLR3 is important for optimal performance of PIKA as an adjuvant. With its good safety profile and antigen-sparing effect, PIKA could be an attractive adjuvant for use in future pandemics. The efficacy of PIKA as prophylaxis against infection with 5 different influenza A virus subtypes was also evaluated in mice. Intranasal treatment with PIKA resulted in significant reduction of viral replication in the respiratory tract. The inhibitory effect was mediated by rapid infiltration of immune cells into the lungs, and production of inflammatory cytokines. While TLR3 is important for the optimal production of these inflammatory cytokines, inhibition of viral replication was still observed in TLR3-/- mice. In addition, a significant synergistic effect in inhibiting H5N1 virus replication was observed when PIKA was co-administered with oseltamivir. The broad-spectrum protection provided by PIKA makes it an attractive option for prophylaxis from infection with influenza A viruses. The influenza virus is constantly changing;immunity to one seasons strains does not translate into immunity in subsequent years. As a consequence, the vaccine is reformulated each year. The neutralizing antibody response to influenza virus is thought to be specific for a few antigenically related isolates within a given subtype. Heterosubtypic antibodies capable of neutralizing multiple subtypes have been recently isolated from phage libraries, but it was not known whether such antibodies are produced in the course of the immune response. In collaboration with scientists at the Institute for Research in Biomedicine in Switzerland, we found that following seasonal influenza vaccination, some individuals produced antibodies that cross-reacted with H5 hemagglutinin (HA). By immortalizing IgG+ B cells from four individuals we isolated a panel of 20 heterosubtypic monoclonal antibodies (mAbs) that bound and neutralized viruses belonging to several HA subtypes (H1, H2, H5, H6 and H9), including the recent pandemic H1N1 A/California/07/09 isolate. The mAbs used different VH genes and carried a high load of somatic mutations. With the exception of a mAb that mapped to the HA globular head, all heterosubtypic mAbs bound to acid sensitive epitopes in the HA stem region. Four mAbs were evaluated in vivo and protected mice from challenge with viruses representative of different subtypes. These findings reveal that seasonal influenza vaccination can induce a polyclonal heterosubtypic neutralizing antibody response that cross-reacts with the swine-origin H1N1 influenza virus and with the highly pathogenic H5N1 virus.